An Ascorbate-induced Absorbance Change in Chloroplasts from Violaxanthin De-epoxidation

Abstract

A new ascorbate-induced chloroplast absorbance change which has the characteristics of a carotenoid shift is described. The absorbance change was light-dependent at pH 7 but not at pH 5. The difference spectra for the light and dark changes were similar, showing a large absorbance peak at 505 nanom-eters, smaller peaks near 468 and 437 nanometers, and a sharp valley around 483 nanometers. The absorbance change is assigned to violaxanthin de-epoxidation because various conditions affected the absorbance change and violaxanthin de-epoxidation similarly, and the difference spectrum resembled the spectrum of zeaxanthin minus violaxanthin in organic solvent. Nigericin with KCI inhibited the light-dependent change at 505 nanometers. This effect, as well as the dark change at pH 5, indicated that de-epoxidation requires an acidic condition in chloroplasts. The effects of 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, 2,6-dichlorophenolindophenol, and phenazine methosulfate indicated that the chloroplast acidification which mediates the 505 nanometers change is derived from hydrogen ion transport linked to photosystem 1. Thus the 505 nanometers change could serve as an endogenous probe for chloroplast acidification and an indirect indicator of hydrogen-ion transport. At pH 5, the role of ascorbate appears to be to provide the reducing potential necessary for reductive de-epoxidation of violaxanthin. At pH 7, ascorbate could have an additional effect of stimulating electron transport and hence the hydrogen-ion transport necessary for de-epoxidation. In contrast to leaves and algae, de-epoxidation in chloroplasts was irreversible under the conditions investigated. Under some conditions other absorbance changes which were apparently due to chlorophyll were superimposed on the de-epoxidation change. The relationship of these ascorbate-induced changes to other absorbance changes observed in chloroplasts and green algae remains to be determined. In leaves, light induces reversible changes between violax-anthin (5,6,5',6'-diepoxyzeaxanthin) and zeaxanthin through the monoepoxide antheraxanthin (17). This conversion, in which epoxide is alternately lost (de-epoxidation) and rein-corporated from 02 (16), appears to be a cyclic pathway for photosynthetic 02 uptake (12) mediated indirectly by pho-1This work was supported in part by National Science Foundation Grant GB8763. This contribution is Journal Series 1331 of the Hawaii Agricultural Experiment Station. tosynthetic electron transport (15). Recently, Hager (5) concluded from the effects of pH and uncouplers that chloroplast acidification from hydrogen-ion transport mediated de-epoxi-dation and that de-epoxidation was therefore related in some way to photophosphorylation. Since zeaxanthin absorbs light at longer wavelengths than violaxanthin, chloroplasts were examined for a difference spectrum which could be correlated with de-epoxidation. We report herein on a new light-induced absorbance change which is stimulated by ascorbate and which has the properties expected of a change from violaxanthin de-epoxidation. A preliminary report on part of this work has appeared (18). MATERIALS AND METHODS Chloroplasts were prepared at 0 C from market lettuce (Lactuca sativa var. Manoa). About 30 g of green leaves were chopped, homogenized in 1 15 ml of SNH solution (0.4 M sorbitol, 10 mm NaCl, 50 mm HEPES, pH 7) for 5 sec in a semi-micro Waring Blendor, filtered through 16 layers of gauze, and centrifuged at 500 or 1OOg for 5 min. The resulting pellet was resuspended in SNH solution and, for spectro-photometric studies, filtered through glass wool to remove large particles. Chloroplast absorbance changes were determined at 25 C with a Perkin-Elmer Model 356 two-wavelength double-beam spectrophotometer. Chloroplast suspensions in 1-cm cuvettes were illuminated with light from a tungsten lamp filtered through red Corning CS2-58 and twoheat-absorbing filters. When required, the photomultiplier was shielded from actinic light with Corning filter CS4-96. Light intensity was measured with a YSI Model 65 radiometer. Violaxanthin de-epoxidation activity in chloroplasts was determined by analysis of changes in pigment composition as described previously (8). The chloroplast suspensions were illuminated in test tubes at 20 C with light from a tungsten-iodine lamp filtered through red cellophane. Chlorophyll concentration was determined according to Vernon (13). RESULTS The light-induced difference spectra of lettuce chloroplasts in the presence of ascorbate both during and after continuous illumination are shown in Figure 1. The spectrum during continuous illumination showed characteristics of a carotenoid shift with peaks at 505, 468, and 437 nm and valleys at 483 and 447 nm. The peak at 437 nm was small and not always evident. After the actinic light was turned off, the spectrum persisted but with a general increase in absorbance below 483 nm. Thus the absorbance change in the presence of ascorbate at 505 nm was not reversible. The partly reversible changes at shorter wavelength appeared to be maximal near 430 nm. which suggested that changes in this re-224 www.plant.org on February 9, 2015-Published by www.plantphysiol.org Downloaded from